As described in the goals and objectives section of this report, this project consists of four specific aims: Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish The development of new tools to facilitate vascular studies in the zebrafish has been an important ongoing aim of this project. In previous work we (i) developed a widely used confocal microangiography method (ii) compiled an atlas of the anatomy of the developing zebrafish vasculature, (iii) generated a variety of transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize vessel formation in intact, living embryos, and (iv) developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish. We are currently developing many new transgenic lines useful for in vivo vascular imaging as well as for in vivo blood or lymphatic endothelial-specific functional manipulation of signaling pathways involved in vascular specification, patterning, and morphogenesis. Notably, we have generated RiboTag zebrafish facilitating high-throughput whole genome profiling of gene expression in blood and lymphatic vascular endothelium and in vascular smooth muscle cells, and we have demonstrated that these transgenic lines can be used toz perform in vivo profiling of vascular signaling pathways. Genetic Analysis of Vascular Development Previously, we have used forward-genetic ENU mutagenesis screens in transgenic zebrafish to generate, identify, and characterize many new zebrafish mutants affecting the formation of the developing vasculature. We identified and positionally cloned mutants with phenotypes including loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. These mutants have resulted in numerous important discoveries related to endothelial specification, arterial differentiation, vascular patterning, and VEGF signaling, to mention only a few. Recent publications include studies on mutants in RECK and aminoacyl-tRNA synthetases affecting VEGF signalling and angiogenesis, on the role of Rac signalling in angiogenesis, and on targeting of phosphoinositide recycling as an anti-angiogenic cancer approach. Most of our current screening efforts are directed at uncovering lymphaticspecific mutants, although we are continuing to obtain and study blood vascular-specific mutants as well. Analysis of Vascular Specification, Patterning, and Morphogenesis We have previously used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and used molecular and experimental analysis understand how this pattern arises and what cues guide vascular specification, differentiation, and network assembly during development. Our discoveries have included evidence that neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning. Our current work includes projects aimed at (a) studying the specification, differentiation, and patterning of vascular smooth muscle in the zebrafish, making use of newly developed transgenic tools, (b) understanding the role of intracellular signaling substrates in regulating vascular endothelial signaling, (c) exploring the role of BMP family ligands in modulating vessel growth and vascular integrity, (d) analyzing additional pro- or anti-angiogenic factors. Emergence of Hematopoietic Stem and Progenitor Cells from the Endothelium We have recently developed an additional aim based on our identification of (i) a novel epigenetic mechanism regulating the emergence of hematopoietic stem and progenitor cells (HSPC) from the endothelium NS (ii) a novel T-cell deficient phenotype in one of our CRISPR mutants. HSPC are critical cells that emerge during early vertebrate development and subsequently seed the development of all blood cell lineages for the life of the animal. We recently reported a novel epigenetic mechanism intersecting with genetic pathways regulating the specification of these cells from endothelium involving maintenance of expression of the key HSPC gene cmyb by gene body methylation by dnmt3bb.1, a DNA methyltransferase specifically expressed in HSPC. We also recently showed that loss of the zap70 gene provides a useful new model for T cell immune deficiency in the zebrafsh.